Fernando Abarzua

Adenovirus-mediated overexpression of REIC/Dkk-3 selectively induces apoptosis in human prostate cancer cells through activation of c-Jun-NH2-kinase

Alteration in genes which takes place during malignant conversion and progression could be potential targets for gene therapy. We previously identified REIC/Dkk-3 as a gene whose expression is reduced in many human cancers. Here, we showed that expression of REIC/Dkk-3 was consistently reduced in human prostate cancer tissues in a stage-dependent manner. Forced expression of REIC/Dkk-3 induced apoptosis in human prostate cancer cell lines lacking endogenous REIC/Dkk-3 expression but not in REIC/Dkk-3-proficient normal prostate epithelial and stromal cells. The apoptosis involved c-Jun-NH2-kinase activation, mitochondrial translocation of Bax, and reduction of Bcl-2. A single injection of an adenovirus vector carrying REIC/Dkk-3 showed a dramatic antitumor effect on a xenotransplanted human prostate cancer. Thus, REIC/Dkk-3 could be a novel target for gene-based therapy of prostate cancer.

Down-regulation of Inhibition of Differentiation-1 via Activation of Activating Transcription Factor 3 and Smad Regulates REIC/Dickkopf-3–Induced Apoptosis

REIC/Dickkopf-3 (Dkk-3), a tumor suppressor gene, has been investigated in gene therapy studies. Our previous study suggested that REIC/Dkk-3-induced apoptosis mainly resulted from phosphorylation of c-Jun-NH(2) kinase (JNK) in prostate cancer cells. However, the precise mechanisms, especially the molecular mechanisms regulating JNK phosphorylation, remain unclear. In this study, we investigated the mechanisms participating in JNK phosphorylation in the context of a refractory cancer disease, malignant mesothelioma (MM). Adenovirus-mediated overexpression of REIC/Dkk-3 induced apoptosis mainly through JNK activation in immortalized MM cells (211H cells). Interestingly, transcriptional down-regulation of inhibition of differentiation-1 (Id-1) was detected in REIC/Dkk-3-overexpressed 211H cells. Moreover, restoration of Id-1 expression antagonized REIC/Dkk-3-induced JNK phosphorylation and apoptosis. Mutagenesis experiments with the 2.1-kb human Id-1 promoter revealed that activating transcription factor 3 (ATF3) and Smad interaction, with their respective binding motifs, was essential for REIC/Dkk-3-mediated suppression of Id-1 promoter activity. ATF3 activation was probably induced by endoplasmic reticulum stress. Finally, we showed strong antitumor effects from REIC/Dkk-3 gene transfer into the pleural cavity in an orthotopic MM mouse model. Relative to control tumor tissue, REIC/Dkk-3-treated tumor tissue showed down-regulated expression of Id-1 mRNA, enhanced expression of phosphorylated JNK, and an increased number of apoptotic cells. In summary, we first showed that both ATF3 and Smad were crucially and synergistically involved in down-regulation of Id-1, which regulated JNK phosphorylation in REIC/Dkk-3-induced apoptosis. Thus, gene therapy with REIC/Dkk-3 may be a promising therapeutic tool for MM.

Adenovirus-mediated REIC/Dkk-3 gene transfer inhibits tumor growth and metastasis in an orthotopic prostate cancer model.

We had previously reported that REIC/Dkk-3, a member of the Dickkopf (Dkk) gene family, works as a tumor suppressor. In this study, we evaluated the therapeutic effects of an intratumoral injection with adenoviral vector encoding REIC/Dkk-3 gene (Ad-REIC) using an orthotopic mouse prostate cancer model of RM-9 cells. We also investigated the in vivo anti-metastatic effect and in vitro anti-invasion effect of Ad-REIC gene delivery. We demonstrated that the Ad-REIC treatment inhibited prostate cancer growth and lymph node metastasis, and prolonged mice survival in the model. These therapeutic responses were consistent with the intratumoral apoptosis induction and in vitro suppression of cell invasion/migration with reduced matrix metalloprotease-2 activity. We thus concluded that in situ Ad-REIC/Dkk-3 gene transfer may be a promising therapeutic intervention modality for the treatment of prostate cancer.

Overexpression of REIC/Dkk-3 in Normal Fibroblasts Suppresses Tumor Growth via Induction of Interleukin-7

We previously showed that the tumor suppressor gene REIC/Dkk-3, when overexpressed by an adenovirus (Ad-REIC), exhibited a dramatic therapeutic effect on human cancers through a mechanism triggered by endoplasmic reticulum stress. Adenovirus vectors show no target cell specificity and thus may elicit unfavorable side effects through infection of normal cells even upon intra-tumoral injection. In this study, we examined possible effects of Ad-REIC on normal cells. We found that infection of normal human fibroblasts (NHF) did not cause apoptosis but induced production of interleukin (IL)-7. The induction was triggered by endoplasmic reticulum stress and mediated through IRE1α, ASK1, p38, and IRF-1. When Ad-REIC-infected NHF were transplanted in a mixture with untreated human prostate cancer cells, the growth of the cancer cells was significantly suppressed. Injection of an IL-7 antibody partially abrogated the suppressive effect of Ad-REIC-infected NHF. These results indicate that Ad-REIC has another arm against human cancer, an indirect host-mediated effect because of overproduction of IL-7 by mis-targeted NHF, in addition to its direct effect on cancer cells.

An N-terminal 78 amino acid truncation of REIC/Dkk-3 effectively induces apoptosis.

Overexpression of REIC/Dkk-3 (a tumor suppressor gene) induces cancer cell apoptosis through endoplasmic reticulum (ER) stress. Therefore, the identification of the portion of REIC/Dkk-3 that causes ER stress may be essential for the development of cancer treatment based on REIC/Dkk-3. Here, we made several truncated forms of REIC/Dkk-3 and investigated their therapeutic potentials against prostate cancer. Among three truncated forms, a variant comprising the N-terminal 78 amino acid region of REIC/Dkk-3 ((1-78)REIC/Dkk-3) most strongly induced ER stress and apoptosis in human prostate cancer cells (PC3). For in vivo gene expression, we coupled a biodegradable polymer with naked DNA, which attained robust trans-gene expression in PC3-derived subcutaneous tumor. In therapeutic experiments, we demonstrated that multiple direct injections of polymer-conjugated (1-78)REIC/Dkk-3 plasmid provoke ER stress and significantly reduced the subcutaneous tumor volume compared with the control group. We suggest this non-viral strategy may be an effective alternative to viral gene therapy.

Intraperitoneal administration of an adenovirus vector carrying REIC/Dkk-3 suppresses peritoneal dissemination of scirrhous gastric carcinoma

Expression levels of the novel tumor suppressor gene REIC/Dkk-3 are reduced in many human cancers. We have previously showed that an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC) induced apoptosis of cancer cells selectively and exerted bystander antitumor effects via ER stress. We examined possible effects of Ad-REIC in a peritoneal dissemination model of scirrhous gastric carcinoma (SGC). Among various types of gastric cancer, SGC continues to be associated with the worst prognosis due to a high incidence of metastases in the peritoneal cavity. We found that a single intraperitoneal injection of Ad-REIC suppressed tumor dissemination and disease progression. Immunomodulation by Ad-REIC led to recruitment of natural killer cells inside tumor nodules. We conclude that Ad-REIC gene therapy may be a potential tool in combinatorial approaches to achieve curative effects in SGC.

Down-regulation of BiP/GRP78 sensitizes resistant prostate cancer cells to gene-therapeutic overexpression of REIC/Dkk-3

We have recently shown that an adenovirus carrying REIC/Dkk‐3 (Ad‐REIC) exhibits a potent tumor‐specific cell‐killing function for various human cancers. It has also become evident that some human cancers are resistant to Ad‐REIC‐induced apoptosis. The aim of the present study was to determine the molecular mechanisms of resistance to Ad‐REIC. First, we isolated resistant clones from a human prostate cancer cell line, PC3, after repeated exposure to Ad‐REIC. Infection efficiency of the adenovirus vector and expression level of REIC/Dkk‐3 in the resistant clones were similar to those in the parental PC3 cells. By screening for alteration in levels and functional status of proteins involved in Ad‐REIC‐induced apoptosis, we found that BiP/GRP78, an ER‐residing chaperone protein, was expressed at higher levels consistently among resistant cells. Expression levels of BiP and rates of apoptosis induced by Ad‐REIC were inversely correlated. Down‐regulation of BiP with siRNA sensitized the resistant cells to Ad‐REIC in vivo as well as in culture. These results indicate that BiP is a major determinant of resistance to Ad‐REIC‐induced apoptosis. Thus BiP is useful for diagnosis of inherent and acquired resistance of cancers and also as a target molecule to overcome resistance to the gene therapeutic Ad‐REIC.

Indications for Ureteropyeloscopy Based on Radiographic Findings and Urine Cytology in Detection of Upper Urinary Tract Carcinoma

OBJECTIVE To verify the indication of diagnostic ureteropyeloscopy based on clinical features for upper urinary tract urothelial cancer with over 100 patients and over a 10-year series. METHODS From January 1997 to December 2008, consecutive 129 units in 124 patients underwent ureteropyeloscopy to obtain a definitive diagnosis of upper urinary tract cancer or to rule out a malignancy. Patients were divided into four subgroups based on voided urine cytology and preoperative radiographic findings: group A (n = 8), positive urine cytology and positive radiographic findings; group B (n = 4), positive cytology and negative radiographic findings; group C (n = 55), negative cytology and positive radiographic findings and group D (n = 62), gross hematuria originating from the upper urinary tract with negative cytology and negative radiographic findings. Ureteropyeloscopic findings were compared with radiographic and cytological results. Adverse effects were also investigated. RESULTS In group A, all patients had confirmed cancer. In group B, one revealed small cancer and the remaining three confirmed carcinoma in situ by biopsy with ureteropyeloscopy. In groups C and D, 33 patients (60%) and four (6.5%) revealed cancer. Seventy-eight patients out of 80 (97.5%) in groups C and D were confirmed to have benign disease. No patient was found with malignancy during follow up after negative finding of ureteropyeloscopy. CONCLUSIONS Ureteropyeloscopy can help in detecting upper urinary tract cancer or to rule out malignancy for patients with negative voiding cytology. However, ureteropyeloscopy is redundant for patients with positive radiographic findings and positive voiding cytology.

Hepatocyte growth factor activator inhibitor-2 (HAI-2) is a favorable prognosis marker and inhibits cell growth through the apoptotic pathway in cervical cancer

BACKGROUND In light of the poor prognosis for cervical cancer, research continues into the development of innovative and efficacious treatment modalities for this disease. We investigated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) and evaluated its clinical importance in cervical cancer. PATIENTS AND METHODS HAI-2 expression was examined in cervical cancer specimens (n=52) by immunohistochemistry. We further attempted to investigate the biological functions and inhibitory effects of HAI-2 using human papillomavirus (HPV) 16 type SiHa and HPV 18 type HeLa cervical cancer cell lines. RESULTS There were significant correlations between HAI-2 expression and stage (P=0.017), lymph node metastasis (P=0.005) and ovarian metastasis (P=0.038). Low HAI-2 expression was a significant predictor for a poor prognosis compared with high HAI-2 expression (disease-free survival rate, P=0.016; overall survival rate, P=0.021). After transient transfection into the SiHa and HeLa cell lines, HAI-2 showed potential inhibitory effects mediated by reductions in hepsin and matriptase expression, which led to apoptosis by increasing the level of Bak and reducing the level of Bcl-2. CONCLUSIONS The present findings indicate that low HAI-2 expression in cervical cancer may be associated with a poor prognosis. We propose that HAI-2 may represent a therapeutic target for the treatment of cervical cancer.

Expression of REIC/Dkk-3 in normal and hyperproliferative epidermis.

Abstract:  Dickkopf (Dkk) family members are known as Wnt modulators involved in the development, cell growth/differentiation and cancer. REIC/Dkk‐3, which does not interfere with Wnt signalling, has been proposed to be a tumor suppressor gene, but its physiological function has remained unclear. In this study, we analysed the expression of REIC/Dkk‐3 in normal interfollicular epidermis (IFE) and hyperproliferative epidermis. REIC/Dkk‐3 was expressed in human and mouse IFE, being localized at the interface of upper spinous layer and granular layer. Skin cancer cell lines lost REIC/Dkk‐3 expression as reported previously. When we analysed patient samples, REIC/Dkk‐3 expression was down‐regulated in the hyperproliferative epidermis including skin cancers and non‐cancerous proliferative diseases. REIC/Dkk‐3 expression was also suppressed in the regenerative and inflammative epidermis of model mice. These findings will certainly contribute to the extension of studies on REIC/Dkk‐3.